Replicative DNA polymerases (pols) copy chromosomal DNA, but stall at lesions caused by DNA damaging agents. Specialized translesion pols have therefore evolved to promote replication past DNA damage thus allowing cells to proliferate in the face of genotoxic agents. Translesion pols are, however, error-prone and thus induce mutations which can lead to tumorigenesis. Pol? is a newly discovered translesion pol which is involved in double-strand break (DSB) repair. Overexpression of pol causes genome instability and high levels of the pol? are found in breast and colorectal cancers, which corresponds to a poor clinical outcome. Reducing the expression level of pol?, however, sensitizes various tumor cells to radiation. Inhibitors of pol? are therefore likely to increase the efficacy and specificity of radiation therapy. Specific Aim 1 will identify and characterize inhibitors of pol? for potential cancer therapeutics. Since translesion pols are error-prone, stringent regulation of these enzymes is necessary for minimizing mutagenesis and cancer risk. How translesion pols are regulated and recruited to sites of DNA damage is poorly understood. Specific Aim 2 will investigate the regulation and recruitment of translesion pol? during translesion synthesis. In addition to bypassing lesions, pol? replicates DNA recombination intermediates called D-loops which are formed during the repair of DSBs. How pol? performs this important function is poorly understood. Specific Aim 3 will investigate the mechanism by which pol? extends D-loop recombination intermediates. Studies perfomed during the mentored stage (Aim 1) will use high-throughput screening and X-ray crystallography to identify and characterize inhibitors of pol? for potential applications in cancer therapeutics. Research performed during the independent stage (Aims 2 and 3) will initially focus on the mechanisms and regulation of human pol? as a model enzyme. These studies will then be extended to pol? which is implicated in the same pathways as pol? such as translesion synthesis and D-loop extension. The proposed research is likely to provide significant insight into the molecular mechanisms of DNA damage tolerance and cancer biology. PUBLIC HEALTH RELEVANCE: Translesion DNA polymerases are specialized molecular machines that copy damaged DNA, which allows cells to proliferate in the face of DNA damaging agents. Mutation and improper regulation of translesion polymerases, however, increases the risk of cancer. Since translesion polymerases are overexpressed in cancers and protect tumor cells from radiation and chemotherapy, investigating the mechanisms and regulation of translesion polymerases is necessary for understanding and treating cancer.